Identification Of Cryptic Species Research Articles

Evolutionary history of Chinese karst loaches (Nemacheilidae, Karstsinnectes): new insights from mitochondrial-based genomes and description of a new species from Guangxi, China

The genus Karstsinnectes of blind fishes known as karst loaches from China was established in 2023 during a revision of the genus Oreonectes (Nemacheilidae). Six species are recorded in this study and some taxonomic progress has been made; however, the lack of genetic data for some species (e.g., K. anophthalmus) may have weakened our current understanding of Karstsinnectes. This study reassessed the phylogeny and evolutionary history of Karstsinnectes by integrating a combination of previously published and newly sequenced mitochondrial genomic data. A phylogenetic tree was developed that was able to divide Karstsinnectes into two clades corresponding to drainages and clarify the phylogenetic position of K. anophthalmus. Divergence times show that Karstsinnectes originated at the Oligocene/Miocene boundary (~22.37 Mya), with the most recent common ancestor occurring in the early Miocene (~18.87 Mya) and interspecific divergence occurring in the late Miocene. Ancestral area reconstruction suggests that the most recent common ancestor of Karstsinnectes most likely inhabited the Hongshui River basin and dispersed into the Zuojiang-Yujiang, Beipanjiang, and Youjiang river basins during the early Miocene (~18.87 Mya), middle Miocene (~12.78 Mya), and late Miocene (~6.71 Mya), respectively. The dispersal under the influence of orogenesis and a monsoon climate drove the speciation and diverse distribution of Karstsinnectes. Such findings are important for conservation considering that Karstsinnectes strictly inhabits deep caves. Additionally, the taxonomic status of the distributed Karstsinnectes population in Leiping Town, Daxin County, Guangxi, China was revised by combining genetic and morphological differences to describe this population as a new species, Karstsinnectes daxinensis Luo, Zhou & Zhou, sp. nov. The definition of the phylogenetic position of K. anophthalmus emphasizes the importance of using type locality material for the identification of cryptic species.

RCR-RFLP approach for identification of cryptic species of Bombus lucorum-complex in Northern European Russia

RCR-RFLP approach for identification of cryptic species of Bombus lucorum-complex in Northern European Russia

Cryptic Diversity of Barred Mudskippers, <i>Periophthalmus argentilineatus</i> (Valenciennes, 1837), from the Southern Coast of Java and East Lombok, Indonesia inferred by <i>COI</i> Mitochondrial Gene

The Barred Mudskipper (P. argentilineatus) is an amphibious fish species that displays fully terrestrial behaviour during low tides. Previous studies have indicated the existence of cryptic species of the barred mudskipper, leading to difficulties in taxonomic identification due to similarities in morphological characteristics. Therefore, this study aimed to generate DNA barcodes for Indonesian barred mudskipper populations. We collected ten specimens from Clungup Beach and Kondang Bandung Beach, representing our samples. Additionally, we incorporated 25 previously collected COI sequences from Indonesia into our analysis. The mitochondrial COI gene was amplified using PCR and analysed using various bioinformatics programs. This study provides evidence for the presence of three genetically distinct clades (A, B, and C) within the P. argentilineatus population in Indonesia, with a deep genetic divergence of 2.41% to 6.12%. Clade A showed a high genetic divergence of 5.51-6.12%, suggesting the presence of a cryptic species consistent with previous studies. The high level of haplotype diversity and low nucleotide diversity observed in each clade suggest a population bottleneck followed by a rapid expansion. The lack of geographical separation in the haplotype network analysis indicates that gene flow between populations may have been facilitated by glaciation events in the past. These findings contribute to a better understanding of the biodiversity of the barred mudskipper species in Indonesia and will aid in the accurate identification of cryptic species. This study highlights the importance of using molecular techniques to complement morphological identification in understanding the evolution and diversity of mudskipper fish species.

Delimiting species, revealing cryptic diversity in Molytinae (Coleoptera: Curculionidae) weevil through DNA barcoding.

The subfamily Molytinae (Coleoptera: Curculionidae), being the second largest group within the family Curculionidae, exhibits a diverse range of hosts and poses a serious threat to agricultural and forestry industries. We used 1,290 cytochrome c oxidase subunit I (COI) barcodes to assess the efficiency of COI barcodes in species differentiation and uncover cryptic species diversity within weevils of Molytinae. The average Kimura 2-parameter distances within species, genus, and subfamily were 2.90%, 11.0%, and 22.26%, respectively, indicating significant genetic differentiation at both levels. Moreover, there exists a considerable degree of overlap between intraspecific (0%-27.50%) and interspecific genetic distances (GDs; 0%-39.30%). The application of Automatic barcode gap discovery, Assemble Species by Automatic Partitioning, Barcode Index Number, Poisson Tree Processes (PTP), Bayesian Poisson Tree Processes (bPTP), and jMOTU resulted in the identification of 279, 275, 494, 322, 320, and 279 molecular operational taxonomic units, respectively. The integration of 6 methods successfully delimited species of Molytinae in 86.6% of all examined morphospecies, surpassing a threshold value of 3% GD (73.0%). A total of 28 morphospecies exhibiting significant intraspecific divergences were assigned to multiple MOTUs, respectively, suggesting the presence of cryptic diversity or population divergence. The identification of cryptic species within certain morphological species in this study necessitates further investigation through comprehensive taxonomic practices in the future.

Development of mitochondrial DNA cytochrome c oxidase subunit I primer sets to construct DNA barcoding library using next-generation sequencing.

Insects are one of the most diverse eukaryotic groups on the planet, with one million or more species present, including those yet undescribed. The DNA barcoding system has been developed, which has aided in the identification of cryptic species and undescribed species. The mitochondrial cytochrome c oxidase I region (mtDNA COI) has been utilised for the barcoding analysis of insect taxa. Thereafter, next-generation sequencing (NGS) technology has been developed, allowing for rapid acquisition of massive amounts of sequence data for genetic analyses. Although NGS-based PCR primers designed to amplify the mtDNA COI region have been developed, their target regions were only a part of COI region and/or there were taxonomic bias for PCR amplification. As the mtDNA COI region is a traditional DNA marker for the DNA barcoding system, modified primers for this region would greatly contribute to taxonomic studies. In this study, we redesigned previously developed PCR primer sets that targetted the mtDNA COI barcoding region to improve amplification efficiency and to enable us to conduct sequencing analysis on NGS. As a result, the redesigned primer sets achieved a high success rate (> 85%) for species examined in this study, covering four insect orders (Coleoptera, Lepidoptera, Orthoptera and Odonata). Thus, by combining the primers with developed primer sets for 12S or 16S rRNA regions, we can conduct more detailed taxonomic, phylogeographic and conservation genetic studies using NGS.

Population genetic structure of Pomacea canaliculata in China based on the COI and ITS1 genes.

Comprehending the phylogeography of invasive organisms enhances our insight into their distribution dynamics, which is instrumental for the development of effective prevention and management strategies. In China, Pomacea canaliculata and Pomacea maculata are the two most widespread and damaging species of the non-native Pomacea spp.. Given this species' rapid spread throughout country, it is urgent to investigate the genetic diversity and structure of its different geographic populations, a task undertaken in the current study using the COI and ITS1 mitochondrial and ribosomal DNA genes, respectively. The result of this study, based on a nationwide systematic survey, a collection of Pomacea spp., and the identification of cryptic species, showed that there is a degree of genetic diversity and differentiation in P. canaliculata, and that all of its variations are mainly due to differences between individuals within different geographical populations. Indeed, this species contains multiple haplotypes, but none of them form a systematic geographical population structure. Furthermore, the COI gene exhibits higher genetic diversity than the ITS1 gene. Our study further clarifies the invasive pathways and dispersal patterns of P. canaliculata in China to provide a theoretical basis.

Morphological Species Delimitation in The Western Pond Turtle (Actinemys): Can Machine Learning Methods Aid in Cryptic Species Identification?

As the discovery of cryptic species has increased in frequency, there has been an interest in whether geometric morphometric data can detect fine-scale patterns of variation that can be used to morphologically diagnose such species. We used a combination of geometric morphometric data and an ensemble of five supervised machine learning methods (MLMs) to investigate whether plastron shape can differentiate two putative cryptic turtle species, Actinemys marmorata and Actinemys pallida. Actinemys has been the focus of considerable research due to its biogeographic distribution and conservation status. Despite this work, reliable morphological diagnoses for its two species are still lacking. We validated our approach on two datasets, one consisting of eight morphologically disparate emydid species, the other consisting of two subspecies of Trachemys (T. scripta scripta, T. scripta elegans). The validation tests returned near-perfect classification rates, demonstrating that plastron shape is an effective means for distinguishing taxonomic groups of emydids via MLMs. In contrast, the same methods did not return high classification rates for a set of alternative phylogeographic and morphological binning schemes in Actinemys. All classification hypotheses performed poorly relative to the validation datasets and no single hypothesis was unequivocally supported for Actinemys. Two hypotheses had machine learning performance that was marginally better than our remaining hypotheses. In both cases, those hypotheses favored a two-species split between A. marmorata and A. pallida specimens, lending tentative morphological support to the hypothesis of two Actinemys species. However, the machine learning results also underscore that Actinemys as a whole has lower levels of plastral variation than other turtles within Emydidae, but the reason for this morphological conservatism is unclear.

Role of Molecular tools in unravelling the cryptic characteristics of termites

Termites, as cryptic organisms, pose significant challenges to traditional methods of species identification and behavior analysis due to their morphological similarities, hidden habitats, and complex social structures. Molecular tools have emerged as transformative techniques for unraveling the cryptic characteristics of termites, aiding in taxonomy, phylogenetics, and population genetics. This research paper explores the pivotal role of molecular approaches, such as DNA barcoding, mitochondrial markers, and genome sequencing, in uncovering termite species diversity and evolutionary relationships. Molecular tools not only enhance the identification of cryptic species but also provide insight into caste differentiation, social behavior, and colony structure. Advanced techniques like transcriptomics and genomics have shed light on termite physiological adaptations, such as cellulose digestion and symbiosis with gut microbes, which are critical for their ecological roles. Additionally, molecular diagnostics offer innovative solutions for termite pest management by identifying genetic markers linked to pesticide resistance and targeting specific species for control measures. This paper reviews key molecular methodologies applied to termites, discusses case studies, and explores future directions in termite research, emphasizing the significance of these tools in understanding termite biology and ecological impact.

TWO LIFE STRATEGIES IN COPEPOD CRYPTIC SPECIES: COEXISTENCE AND DISPLACEMENT

Among invasive species there is a special group so named cryptic species for which morphological identification is very difficult. This review is devoted to analysis of the dispersal routes of two copepod cryptic species complexes into aquatic ecosystems. Eurytemora carolleeae introduction was revealed in 2007 with bar-code. The species was described as a new taxon; its distribution was also studied using morphology. Biological invasions of two other Copepod species Acanthocyclops americanus and Eurytemora caspica were mainly studied using morphological methods since the species have already been described. At the same time, to confirm their distinctions from local forms, molecular genetic tools were also used. Two scenarios resulting from cryptic species’ invasions and their competitions with native species were the partly ( E. carrolleeae ) or full displacement ( A. americanus ). Example: the invasion of Eurytemora carolleeae into the Baltic Sea and the Atlantic coast of Europe. When assessing the possible negative impact of invasive species on competitors, the most attention should be paid to predator species. Identification of cryptic species significantly complicates the situation with the assessment of bioinvasion and needs the use of molecular methods.

Next‐generation sequencing reveals hidden genomic diversity in glacial relicts: A case study of Meesia triquetra

AbstractThe recent development of genetic methods has facilitated the identification of cryptic species across different groups of organisms, including plants. However, next‐generation sequencing has rarely been used to study cryptic speciation in plants, especially in bryophytes, organisms with a dominant haploid life phase. The ability to capture variation across the whole genome makes this method an effective tool for distinguishing cryptic lineages. We have focused on the genetic structure of the moss Meesia triquetra along the Alps‐to‐Scandinavia transect. We detected the presence of the two genetically critically different lineages of M. triquetra in Europe. These lineages overlap in both morphological characters of the gametophyte and distribution ranges. However, they considerably differ in ecological preferences to groundwater pH. While lineage 1 occupied alkaline to subneutral fens, lineage 2 occurred in fens saturated with neutral to acidic water. We consider the entities cryptic species with respect to genetic and ecological differences but the absence of morphological features necessary for determining the entities. We hypothesize that fragmentation of the ancestral population of the moss in geographically isolated refugia differing in the commonness of acidic and alkaline substrates led to consequent long‐term adaptation to different environmental conditions, then drove diversification in M. triquetra.

Genetic Identification of Two Mudskipper Species (Oxudercidae: <i>Periophthalmus</i>) from Kulon Progo, Special Region of Yogyakarta, Indonesia

Mudskippers are commonly cryptic species, making identification based solely on morphological characteristics challenging. This study used the DNA barcoding method to identify mudskipper species based on the COI mitochondrial gene. The analysis revealed two distinct species, P. kalolo (20 samples) and P. argentilineatus (3 samples) with high GC contents ranging from 42.94-45.2%. The genetic divergence analysis of P. kalolo showed that they divided into two clades, while P. argentilineatus is divided into three clades with two of the clades (C and D) still conspecific groups, and those two clades with clade E exhibit a genetic distance greater than 3.5%, suggesting the presence of cryptic species. These findings provide valuable insights into the intraspecies genetic diversity of mudskippers in Indonesia, which could have essential implications for conservation efforts and highlight the potential of DNA barcoding as a powerful tool for the identification of cryptic species. Further research combining molecular and morphological identification could lead to a more comprehensive understanding of species identification and help address the challenges posed by cryptic species.

Mitochondrial COI based molecular identification of harvester termite, Anacanthotermes ochraceus (Burmeister, 1839) in Riyadh Region, the Kingdom of Saudi Arabia

ObjectiveTermites are well known for being the most destructive pests of household commodities as well as agricultural crops around the globe. The termite fauna (Isoptera) has about 2650 described species worldwide. Several species are the pests of crops and cause damage to wood structures. MethodsIn the present study, 29 specimens of termites collected from different localities of the Riyadh region were identified using mitochondrial gene cytochrome c oxidase subunit I (COI) deoxyribonucleic acid (DNA) sequence. COI gene was PCR amplified using universal primers (LCO 1490 and HCO 2198). MEGA7 software was used for phylogenetic tree construction which showed that all 29 specimens grouped together in a single clade indicated close relatedness of all specimens. ResultsAll the obtained sequences were submitted into Genbank database and accession numbers were obtained. Phylogenetic analysis showed that all specimens of present research grouped together into a single monophyletic clade, were confirmed to be highly closely related to one another, and proved to be members of the same species. Pairwise nucleotide sequence divergence analysis showed that there was less divergence among all specimens ranging from 0% to 7.8%. Sequence analysis revealed the confirmed precise identification of 29 samples of Anacanthotermes ochraceus with COI barcode analysis. ConclusionsMolecular data analysis has confirmed morphological identification of all 29 studied samples of A. ochraceus. However, this technology offers strong support for identification of cryptic species which are difficult to identify on the basis of morphological features. Further studies of complete mitogenome can be helpful for accurate identification of termites at species level.

Genetic Diversity and Cryptic Species Identification of Genus Triplophysa from River Swat in Malakand Division, Khyber Pakhtunkhwa, Pakistan

Fish are cold blooded vertebrates’ identification on the bases of morphology is not more precise and required high taxonomic expertise therefore molecular identification is used as an alternative and more accurate technique for the identification of fishes. In the current investigation 39 fish specimens were collected from May 2021 to February 2022. With this aim, the recent study was conducted in the freshwaters of River Swat in Malakand Division, Khyber Pakhtunkhwa, Pakistan to explore the genetic diversity of genus Triplophysa. DNA was extracted and amplified using gene specific primers. The PCR products carefully were sequenced and Phylogenic analysis was performed using neighbor-joining, maximum likelihood through MEGA software. Nucleotide composition and genetic pairwise distance recommend that the calculated species of the subfamily Nemacheilinae are organically dissimilar. The River Swat is the study area and it is inhabited by three Triplophysa species, Triplophysa naziri, Triplophysa microps, and Triplophysa choprai. The evolutionary tree shows that these species are clearly separated. The mean of total length calculated in the three species of the genus Triplophysa such as 8.175 ± 0.198 cm for Triplophysa naziri, 10.14 ± 0.35 cm for Triplophysa microps, 11.052 ± 0.23 cm for Triplophysa choprai. This study provides a model for the improvement of identification in cryptic diversity and field of ichthyofauna.

USING SHORT SEQUENCE matK GENE AS BARCODE DNA FOR IDENTIFICATION OF DURIO Sp IN TERNATE ISLAND

The Barcode of Life Consortium (CBOL) recommended a standard method for the identification of plant species using matK and rbcL barcoding gene. This study was aimed to evaluate the efficiency of matK DNA barcoding for identification of local durian (Durio sp.) from Ternate Island. Total 15 local durian has been used in this study. Whole genom DNA was isolated by Geneid plant DNA kit and then successfully amplified by the Polymerase Chain Reaction (PCR) technique using specific primer. MatK successfully amplified with 245 bp in length. MatK has sequencing success (71.3%) and relative high of Quality value 20+ (86%). BLAST analysis of the sequence showed that local durian in Ternate are identifies as Durio zibethinus and Neesia malayana with query cover 97%-99%. It could be concluded that matK with short sequence is not efficience for durian identification. The recommended in this studies for using of molecular markers with sequence lengths above 500 bp would be more effective for the identification of cryptic species Durio sp. and other.

Calcification traits for cryptic species identification: Insights into coralline biomineralization.

Calcareous red algae are foundation species and ecosystem engineers with a global distribution. The principles governing their calcification pathways are still debated and the morphological characters are frequently unreliable for species segregation, as shown by molecular genetics. The recent description of the new species Lithophyllum pseudoracemus, previously undetected and morphologically confused with Lithophyllum racemus, offered a challenging opportunity to test the effectiveness of microanatomy and ultrastructural calcification traits as tools for the identification of these two species, for integrative taxonomy. High resolution SEM images of molecularly identified samples showed that the different size of the perithallial cells and the features of the asexual conceptacle chambers may contribute to the separation of the two species. The two species share the same crystallite morphology in the primary and secondary cell-wall calcification, as previously described in other species belonging to the same clade. However, the perithallial secondary calcification was significantly thicker in L. racemus than in L. pseudoracemus. We described a granular calcified layer in the innermost part of the cell wall, as a putative precursor phase in the biomineralization and formation of the secondary calcification. The hypothesis of different pathways for the formation of the primary and secondary calcification is supported by the observed cell elongation associated with thicker and higher Mg/Ca primary calcification, the inverse correlation of primary and secondary calcification thickness, and the absence of primary calcification in the newly formed wall cutting off an epithallial cell from the meristem.

P401 Identification and characterization of cryptic species of Aspergillus isolated from clinical samples

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM Background and ObjectiveMolds are emerging as a major cause of life-threatening infections in immunocompromised patients. There is an increasing recognition of the cryptic Aspergillus species, which are organisms that are morphologically indistinguishable yet can be differentiated by molecular methods. These organisms have been known to show a higher minimal inhibitory concentration for the majority of the antifungal agents in vitro. Therefore, correct identification of these cryptic species is very important to administer a proper antifungal agent.In this study, we wish to identify and characterize the cryptic species of Aspergillus from all clinical samples.MethodsPatient routine samples like broncho alveolar lavage, endo-tracheal aspirate, sputum, pus, tissue, and CSF from various wards, OPDs, and ICUs of All India Institute of Medical Sciences, New Delhi which were processed in the Mycology laboratory, department of Microbiology showing growth of Aspergillus species were included in this study. Identification of the isolates was done using phenotypic methods and by Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF). For the analysis of cryptic species, the isolates underwent PCR and then sequencing of the ß-tubulin gene was done. Antifungal susceptibility testing was done using micro broth dilution as per the CLSI method.ResultsOf the 92 isolates, using morphological methods, 53 were identified as A. fumigatus, 33 were identified as A. flavus, 3 as A. nidulans, 2 as A. terreus, and 1 as A. niger MALDI-TOF (Vitek MS database) misidentified 2 isolates of A. nidulans and 1 isolate of A, stellatus as A. fumigatus and 1 isolate of A. tubingensis as A. niger. The ß-tubulin sequence analysis for the identification of cryptic species revealed that 2 isolates (2.08%) were cryptic, one was A. stellatus morphologically identified as A. nidulans and another one was A. tubingensis morphologically identified as A. niger.ConclusionCurrently the available data on cryptic Aspergillus species is very limited. Because of its varied susceptibility pattern, it is important to identify the Aspergillus isolates to its species level. There is also a need for expansion of the number of strains for each species in MALDI-TOF MS databases for convenient, faster, and correct identification.

P455 Identification of Cryptic species of Aspergillus using Beta-tubulin gene in a tertiary care center in South India

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesTo identify cryptic species of the genus Aspergillus using Beta-tubulin gene by sequence typing.Methods Aspergillus grown from various clinical samples (Ear swab, Bronchial wash, Endotracheal Aspiration, Paranasal sinus, BAL, Sputum) was subcultured on Sabouraud's Dextrose Agar/Oatmeal Agar.Tease mount/Slide culture was done to study the morphological features of the hyphae, size, shape, and arrangement of the conidia.DNA Extraction using phenol-chloroform method: DNA was extracted and purified directly from 4-day-old culture and used as a template for polymerase chain reaction (PCR) amplification.DNA Amplification: A fragment of the target gene were amplified using PCR.The primer pairs used for Beta-tubulin gene was Bt2a (Forward primer) and Bt2b (Reverse primer).Beta-tubulin.Bt2a FGGTAACCAAATCGGTGCTGCTTTCBt2b RACCCTCAGTGTAGTGACCCTTGGCDNA Purification: The PCR product was purified with multi-screen filter plates.Gel electrophoresis: The amplified DNA product was subjected to agarose gel electrophoresis and specific band formation was to be observed for the species of Aspergillus.DNA Sequencing: The purified product will be used as a template for sequencing. An applied biosystem 3730 sequencer will be used to obtain DNA sequences.Phylogenetic Analysis: Parsimony analysis of individual and combined matrices will be conducted using PAUP version 4.0b10 software/free software at phylogeny.fr by the maximum parsimony (MP) and Neighbor-joining methods.The results will be represented using a dendrogram.ResultsA total of 30 Aspergillus isolates were collected and identified using Phenotypic methods.The DNA extraction and PCR amplification were done and sent for Sequencing. The results of Sequence typing are awaited.ConclusionCryptic species are morphologically indistinguishable forms of Aspergillus and their identifications can be confirmed exclusively by using molecular techniques which have led to the description of previously unknown or rare species among different Aspergillus species complex. According to studies, the frequency of such species was found to be 10% in TRANSNET study U.S and 12% in FILPOP study Spain. Most of these species are less susceptible and certain species are multidrug-resistant (azoles and amphotericin B) and the emergence of antifungal-resistance among the Aspergillus species forms the importance in identifying Cryptic species.

Attack on crypsis: Molecular and morphological study of Dendrodoris Ehrenberg, 1831 (Mollusca: Gastropoda: Nudibranchia) from the Mediterranean Sea and Northern Atlantic Ocean reinstates Dendrodoris temarana Pruvot-Fol, 1953.

The genus Dendrodoris is ascribed to the family Dendrodorididae, whose members are characterized lacking spicules in the dorsum as well as a radula, one of the most important taxonomic traits to distinguish and define nudibranch species. The absence of this taxonomic character makes difficult to delineate species based on morphologic traits, and consequently, several cryptic species may remain unreported resulting in a potential underestimation of the real diversity of the genus. Species delimitation analyses based on molecular data constitute an important tool to unveil cryptic species, especially when morphology may be misleading. However, for the genus Dendrodoris, only two molecular studies have been performed to date, both based on Indo-pacific species and the molecular marker COI. In the present study, we performed a multilocus approach using the molecular markers COI, 16S and H3 based on 47 Dendrodoris specimens from the North Atlantic Ocean and Mediterranean Sea. As a result, we detected monophyly for five of the previously accepted species plus the presence of an unidentified one. Using morphological and anatomical data from 14 additional individuals and a review of the literature, we verified this unidentified species is the previously synonymized species Dendrodoris temarana Pruvot-Fol, 1953, and we redescribe it morphologically (chromatic pattern, gills, rhinophores and anal papilla), anatomically (neural, circulatory, digestive and reproductive systems), and biologically herein. D. temarana is differentiated by its chromatic pattern, previously thought to be intraspecific variation of D. grandiflora (Rapp, 1827), D. limbata (Cuvier, 1804) and D. herytra Valds Ortea in Valds, Ortea, Avila Ballesteros, 1996, and therefore it is considered a pseudocryptic species. This study increases the number of valid species in the North Atlantic and Mediterranean to nine and confirms the importance of integrative taxonomic approaches to resolve the identification of cryptic species in complex groups.

Cryptic Species Diversity and Phylogenetic Relationship in the Rust Genus Chrysomyxa from China.

Chrysomyxa rusts are fungal pathogens widely distributed in the Northern hemisphere, causing spruce needle and cone rust diseases, and they are responsible for significant economic losses in China. Taxonomic delimitation and precise species identification are difficult within this genus because some characters often overlap in several species. Adequate species delimitation, enhanced by the use of DNA-based methodologies, will help to establish well-supported species boundaries and enable the identification of cryptic species. Here, we explore the cryptic species diversity in the rust genus Chrysomyxa from China. Species delimitation analyses are conducted using a distance-based method (ABGD) and three tree-based methods (GMYC, bPTP, and mPTP) based on combined LSU and ITS sequences of over 60 specimens. Although there is some incongruence among species delimitation methods, two new species and three putative cryptic species are identified. The key to 20 Chrysomyxa species distributed in China is presented. These results suggest that a significant level of undiscovered cryptic diversity is likely to be found in Chrysomyxa from China. Future studies should consider multiple analytical methods when dealing with multi-locus datasets.

Are members of the Anopheles fluviatilis complex conspecific?

Anopheles fluviatilis sensu lato, a primary malaria vector in India, has been identified to be comprised of four cryptic species, provisionally designated as species S, T, U and V. However, Kumar et al. (Mol Ecol Resour, 2013;13:354-61) considered all of the then known three members of this species complex (S, T and U) conspecific. The specific status of species S and T was refuted based on the lack of sufficient barcode gap in mitochondrial-CO1 and the perceived presence of heterozygotes in populations as detected through one of the two species-specific PCR assays employed for the cryptic species identification. The existence of species U was refuted claiming that earlier investigations have already refuted their existence. Here we discuss problems associated with the CO1-based barcode approach for delimitation of cryptic species, the perceived heterozygosity between species S and T based on a species-specific PCR assay, and interpretation of published reports. We demonstrated that fixed differences do exist in the ITS2-rDNA sequence of species S and T with no evidence of heterozygotes in sympatric populations and, that the observed heterozygosity by Kumar et al. in the ITS2-based species diagnostic PCR is due to the high mispriming tendency of the T-specific primer with species S. We infer that mitochondrial DNA-based ‘barcoding gap’, an arbitrary threshold recommended for species delimitation, alone, is inadequate to delimit the members of An. fluviatilis complex.